DNA Repair Mechanisms PDF - Quest International University - 12/2024

MM 6113 BL 4 DNA repair mechanisms Dr. Koh Cai Ping 12/2024 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  LEARNING OUTCOMES At the end of this session, students would be able to Explain the role of telomeres and telomerase in replication p...

MM 6113 BL 4 DNA repair mechanisms Dr. Koh Cai Ping 12/2024 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  LEARNING OUTCOMES At the end of this session, students would be able to Explain the role of telomeres and telomerase in replication process v Describe DNA repair mechanisms o Base Excision Repair o Nucleotide Excision Repair o Mismatch Repair o Direct Repair (Photo reactivation Repair) o Double Strand Break Repair 2 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Telomeres shorten with each cell division. When the telomere is shortened beyond some critical length cell is no longer able to divide → apoptosis v Senescent or aging In germ cells and some cancer cells Telomere is protected from shortening Due to the presence of telomerase Keep dividing → immortal 30-Dec-24 3 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  TELOMERE AND TELOMERASE v Elizabeth Blackburn, 2009 Nobel Laureate, is the scientist who discovered how telomerase Key finding: telomerase replenish telomere works. (credit: US Embassy Sweden) 4 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Telomere Complex of repetitive DNA + proteins Located at the end of chromosomes Source: Wikipedia Structure v http://research.physics.berk Tandem repeats of TTAGGG eley.edu/yildiz/SubPages/res earch_telomeres.html The GT-rich strand is longer → ssDNA ~ a few hundred nucleotides in length at the 3'-end The ssDNA region is folded and forms a loop structure that is stabilized by protein Telomeric Repeat Factor 1(TFR1) and TRF2 – is a protein that binds to telomere ends o Maintain telomere length 5 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Telomere Functions Maintain the structural integrity of the chromosome Protect DNA from nucleases activities Distinguish a true end from a break in dsDNA (repair mechanism) v During DNA replication Removal of RNA primer causes shortening of telomere at the 5’ end of the newly synthesized DNA strand After development, cells stop synthesizing telomerase Shortening of telomere Eventually age 6 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Telomerase multi-subunit ribonucleoprotein Reverse transcriptase that contains a short sequence of RNA v that acts as a template To maintain the telomere length 7 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  The role of telomerase in diseases Age-related disease Cardiovascular disease (CVD), type 2 diabetes, Alzherimer’s disease, osteoporosis. In cancer cells v Telomerase expression is induced Maintain the length of telomere→ skip aging Uncontrolled cell growth https://www.mdpi.com/2227- 9059/9/10/1335#:~:text=Because%20of%20their%20protective%20function%20for%20genomic%20DNA%2C,as%20cardiovascular% 20disease%20%28CVD%29%2C%20malignancies%2C%20dementia%2C%20and%20osteoporosis. 8 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my DNA repair mechanisms v Thymidine dimer 9 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Types of DNA damages Mismatch of bases e.g. A bind G Dimer formation e.g. Thymine dimer v Bases alterations (abnormal bases) Deamination of cytosine Substitution of nucleotides Single strand breaks Double strand breaks QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  DNA repair mechanism Principles 1. Recognize Identify types of damage 2. Remove v 3. Re-synthesize DNA polymerase 4. Re-ligate DNA ligase 11 DNA repair mechanisms 1. Mismatch repair (MMR) 2. Nucleotide excision repair (NER) 3. Base excision repairv (BER) 4. Direct repair (photo reactivation repair) 5. Double-strand break repair 12 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  1. Mismatch repair DNA damage: mismatch during DNA replication Repair mechanism: v 1. Recognize Methylation of adenine residue in GATC sequence in a parental DNA strand Newly synthesized DNA strand does not undergoes methylation immediately 13 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  1. Mismatch repair MutS, MutL and MutH proteins (prokaryotes) hMLH1, hMSH2, hPMS1 and hPMS2 (human) 2. Remove v mismatch nucleotide is removed 3. Re-synthesis DNA polymerase 4. Re-ligate DNA ligase 14 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my Mechanism of MutS, MutH & MutL MutS: recognized the mismatch (heteroduplex) MutL: a dimer; in presence of ATP, binds MutS-heteroduplex complex to activate MutH MutH: endonuclease that cleaves 5’ to the G in an unmethylated GATC, leaves a nick. 15  1. Mismatch repair 1. Lynch syndrome Mutations of MLH1, MSH2, MSH6, and PMS2. Also known as hereditary nonpolyposis colorectal cancer (HNPCC). Increased risk of colorectal cancer, endometrial v cancer and other malignancies. Diagnostic methods: Patient history, genetic testing, and immunohistochemistry (IHC). Clinical features: Blood in stool, constipation, etc. 16 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  2. Nucleotide Excision Repair DNA damage: thymine dimer caused Prokaryotic system by UV light exposure Repair mechanism: 1. Recognized and excised by UV- v specific endonuclease UvrABC excinuclease Identify and cleave thymine dimer 2. Re-synthesis and re-ligation QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my 2. Nucleotide Excision Repair Eukaryotic system GGR After lesion recognition by XPC, the ATPase activity of XPB recruits the holo TFIIH to damaged DNA and the helicase activity of XPD opens the DNA around the lesion. Subsequently, the downstream NER v factors XPA, XPG, and ERCC1-XPF trigger excision of the damaged oligonucleotide. TCR TC-NER initiates when RNA polymerase stalls at a lesion in DNA: the blocked RNA polymerase serves as a damage recognition signal, which replaces the need for the distortion recognition properties of the XPC-RAD23B and DDB complexes. CS proteins (CSA and CSB) bind some types of DNA damage instead of XPC-Rad23B. QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  2. Nucleotide Excision Repair 1. Xeroderma pigmentosum (XP) Mutations of XPB, XPD, XPG etc genes involved in GG-NER. Common in Japan, North Africa, and the Middle East. Autosomal recessive inheritance v Diagnostic methods: Self-check, physical examination, laboratory test. Clinical features: Skin: Lentigos, xerosis (dry, parchment like skin) and poikiloderma a mixture of both hyper (increased) or hypo (decreased) skin pigmentation. Eye: Photophobia (light sensitivity, or pain upon seeing light) Nervous system: around 25% developed progressive neurodegeneration. Sensitive to UV light → skin cancer by age 10 (without treatment） 19 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  2. Nucleotide Excision Repair 2. Cockayne syndrome (CS) Caused by changes (pathogenic variants) in the ERCC6/CSB (65%) and ERCC8/CSA (35%) genes > TC-NER Autosomal recessive CS classification: i. Classical Form, Cockayne Syndrome Type I (Type A) – onset after the age of 1 ii. Congenital Form, Cockayne Syndrome Type II (Type v B) – onset at birth iii. Late Onset, Cockayne Syndrome Type III (Type C) Diagnostic methods: Molecular genetic testing for pathogenic variants. DNA repair assay on skin cells (UV test). Clinical features: Short stature Premature aging (progeria) Severe photosensitivity Microcephaly Death typically occurs by the age of 12. 20 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  2. Nucleotide Excision Repair 3. Trichothiodystrophy (TTD) Mutations in the GG-NER repair genes XPD (95%), XPB or TTDA. Autosomal recessive Diagnostic methods: Trichoschisis (broken or split hairs) v A severely damaged or absent hair cuticle by electron microscopy. Clinical features: Sulphur-deficient, short, brittle hair (easily broken) Intellectual impairment or developmental delay (85% of cases) Short stature (73% of cases) Ocular abnormalities (50% of cases) Photosensitivity (but no confer risk of skin cancer) https://dermnetnz.org/topics/trichothiodystrophy#:~:text=%20What%20causes%20trichothiodystrophy%3F%20%201%20Photosensitive%20trichothiodystr ophy,%28%20TTDN1%20%29%2C%20a%20gene%20of...%20More%20 21 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  3. Base Excision Repair DNA damage: base alteration, eg: deamination (hydrolysis of bases) of cytosine → uracil (spontaneously) v Repair mechanism: 1. Nitrogenous base is recognized & cleaved by Uracil N-glycosylase Cleaves the bond between the base and the pentose sugar 22 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  3. Base Excision Repair Uracil is removed, but left the deoxyribose-phosphate backbone → referred as apyrimidinic site ( AP-site) v 2. Removal of AP-site o AP-endonuclease recognizes & nicks o Deoxyribose-phosphate lyase remove the empty deoxyribose phosphate residue 23 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  3. Base Excision Repair 3. Re-synthesis o DNA polymerase v 4. Re-ligation o DNA ligase 24 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  4. Direct repair (Photoreactivation repair) DNA damage: Thymine dimer Energy from visible light is used to break the dimers Enzyme: DNA photolyase v Common to prokaryotic and some eukaryotic cells Not universal Does not exist in human 25 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  4. Direct repair (Alkylating agents) DNA damage: O6 – methylguanine - alkylation of bases (usually methylation) Repair mechanism: - Recognized and removed by O6 –v methylguanine methyltransferase Transfer methyl group onto itself Restore guanine base Occurs in prokaryotes and eukaryotes (including human) 26 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  5. Double-strand breaks repair DNA damage: double-strand break A. Non-homologous end-joining repair (NHEJ) v Two DNA fragments are joined Regardless to sequence homology Mutagenic → cancer B. Homologous recombination repair (HR) 27 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  NHEJ Do not require homologous chromosome 1. Ku80/70 and DNA-Protein kinase complex binds to the end of each DNA break point 2. Remove nucleotides 3. Ligation Effects: few bases are removed → mutation - May accidentally fuse two different chromosomes v HR 1. Assembly of repair protein 2. Resection of DNA 3. Strand invasion 4. Use homologous DNA as a template to repair the break 5. The double stranded DNA break is repaired with a high level of fidelity QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  5. Double-strand breaks repair A. Non-homologous end joining repair Do not require homologous chromosome 1. Ku80/70 and DNA-Protein kinase complex binds to the end of each DNA break point v 2. Remove nucleotides 3. Ligation Effects: few bases are removed → mutation - May accidentally fuse two different chromosomes 29 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  5. Double-strand breaks repair B. Homologous recombinant DNA repair (require homologous chromosome) 1. Assembly of repair protein 2. Resection of DNA v 3. Strand invasion 4. Use homologous DNA as a template to repair the break 5. The double stranded DNA break is repaired with a high level of fidelity Image credit: Buddhini Samarasinghe 30 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Disorders related to DNA replication and repair mechanism 4. Ataxia Telangiectasia disease The protein kinase Ataxia-telangiectasia mutated (ATM) is at the heart of the cellular response to double-stranded break DNA damage, homologous repair (HR). Autosomal recessive neurodegeneration v Diagnostic methods: Self-check, laboratory test, imaging. Clinical features: Usually developed before age 5. Difficulty with coordinating movement (ataxia). Oculomotor apraxia Progressive cerebellar ataxia Choreoathetosis QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Disorders related to DNA replication and repair mechanism 5. Bloom’s syndrome Mutations in the BLM gene which encode RecQ helicase Particularly prevalent in people with Ashkenazi Jewish ancestry (Jews from northern and eastern Europe) Autosomal recessive v Diagnostic methods: Blood testing Bloom’s syndrome Genetic testing Prenatal testing - Ultrasound Clinical features: Abnormal growth (usually not surpass 5 feet tall after reaching adulthood) Butterfly-shaped patch of reddened skin across the nose and cheeks. Long, narrow face, a smaller lower jaw or chin, pronounced nose, and prominent ears. Photosensitivity Infertility in men 32 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  Disorders related to DNA replication and repair mechanism 6. Fanconi Anemia Defective repair of DNA interstrand crosslinks (ICLs). enlists elements of three classic DNA repair pathways, namely homologous recombination, nucleotide excision repair, and mutagenic translesion synthesis, in response to genotoxic insults. Chromosomal breakage (CB) test. Autosomal recessive v Diagnostic methods: Physical Examination Chromosomal breakage (CB) test. Imagning Clinical features: Half the patients are diagnosed prior to age 10. Bone marrow failure Thumb hyperplasia café au lait spots (coffee color spots on skin) By the age of 40, 98% of FA patients will have developed some type of hematological abnormality. Increased risk of acute myeloid leukemia (AML), solid tumors & aplastic anemia. QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my  v Digweed, 1993 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my Targeting DNA repair in cancer treatment v QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my Targeting DNA repair cancer treatment v Beggs and Yang, 2019 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my References Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. DNA Repair. Available from: http://www.ncbi.nlm.nih.gov/books/NBK9900/Your text Richard Harvey and Denise Ferrrier. 2011. Biochemistry. (5th edition). Lippincott Williams v & Wilkins. Mismatch repair https://www.youtube.com/watch?v=p3MXIKWAi2w The Key to Chromosome Immortality - iBiology & Youreka Science https://www.youtube.com/watch?v=f- JLnfnGPyA 37 QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my THANK YOU QUEST INTERNATIONAL UNIVERSITY (DU021(A)) | www.qiu.edu.my